The proliferation and differentiation of cells in multicellular organisms is subject to a highly regulated process. A distinguishing feature of cancer cells is the absence of control over this process; proliferation and differentiation become deregulated resulting in uncontrolled growth. Significant research efforts have been directed toward better understanding this difference between normal and tumor cells. One area of research focus is growth factors and, more specifically, autocrine growth stimulation.
Growth factors are polypeptides which carry messages to cells concerning growth, differentiation, migration and gene expression. Typically, growth factors are produced in one cell and act on another cell to stimulate proliferation. However, certain malignant cells, in culture, demonstrate a greater or absolute reliance on an autocrine growth mechanism. Malignant cells which observe this autocrine behavior circumvent the regulation of growth factor production by other cells and are therefore unregulated in their growth.
B cell development is composed of two phases: antigen independent and antigen dependent. The antigen-independent phase of B cell development occurs in the bone marrow where B cell progenitors differentiate into immature B cells expressing cell surface IgM. The antigen-dependent phase of B cells differentiation occurs in the peripheral secondary lymphoid organs where antigen-specific B cells proliferate and differentiate into plasma cells that secrete specific antibody upon activation.
During the antigen-independent phase of B cell development, sequential rearrangement of immunoglobulin gene segments generates a diverse repertoire of antigens. Pro-B cells, the earliest B-lineage cells derived from B cell progenitors, are characterized by the appearance of early B-cell lineage cell-surface proteins and by immunoglobulin gene rearrangement of the heavy-chain locus. The pro-B cell stage is followed by pre-B cell stage which is characterized by the rearrangement of the immunoglobulin light chain gene. Successful rearrangement of both heavy and light chains leads to the expression of intact IgM molecules on the cell surface at the immature B cell stage.
Immature B cells undergo selection for self-tolerance in a series of checkpoints triggered by antigens and selection for the ability to survive in the peripheral lymphoid tissues. B cells that survive the selection for self-tolerance and the ability to survive in the peripheral lymphoid tissues further differentiate to become mature B cells that express surface IgD in addition to surface IgM [Burrows, 1997]. Mature B cells recirculate through peripheral lymphoid tissues where they may encounter antigens. B cells activated by antigen may differentiate into plasma cells and secrete a large amount of antibodies [Duchosal, 1997]. There are 5 different classes of immunoglobulin molecule: IgM, IgD, IgG, IgA, and IgE. IgM is the first immunoglobulin molecule to be synthesized and expressed.
Antigen dependent B cell development and differentiation begin with the binding of antigens on B cells. B cell activation requires two signals: binding of the antigen to the B-cell surface immunoglobulin and interaction of B cells with antigen-specific helper T cells. The surface immunoglobulin serving as the B-cell antigen receptor (BCR) has an important role in B-cell activation. After binding the antigen, the BCR and antigen complex is internalized and the antigen protein is degraded. The digested antigen returns to the B-cell surface as peptides bound to MHC class II molecules [Parker, 1993].
As B cells develop from pro B cells to plasma cells, they express cell surface proteins other than immunoglobulin that are useful markers for B-lineage cells at different developmental stages. One of the first identifiable proteins expressed on the surface of B-lineage cells is CD45R (also known as B220) [Osmond, 1998; Hardy, 2001]. CD45R, a protein tyrosine phosphatase that functions in B-cell receptor signaling, is expressed throughout B-cell development from pro-B cells right up to plasma cells [Osmond, 1998; Hardy, 2001]. CD43 (the mucin leukosialin) is also expressed at the pro-B cell stage but its expression is lost as cells progress to immature B cells [Hardy, 2001]. CD43 is a multi-functional molecule with directly contradictory functionality [Ostberg, 1998]. For example, CD43 can act as an adhesion molecule that may guide cell-cell interactions of B-cell precursors with stromal cells [Ostberg, 1998]. However, CD43 also has anti-adhesion functions [Ostberg, 1998]. CD43 has an important role in cell signaling and cytoskeletal interaction [Ostberg, 1998]. CD19 is another surface marker protein expressed from pro-B cells through the plasma cell stage [Hardy, 2001]. CD19 is involved in B-cell receptor signaling and lowers the threshold for antigen receptor stimulation of B cells [LeBien, 1998]. Other cell-surface molecules expressed during different stages of B-cell development include the heat-stable antigen HSA, CD 10, CD 20, CD 22, CD38, and CD40 [Duchosal, 1997; Hardy, 2001].
B cell development and differentiation of antigen independent phase are tightly regulated by lineage and stage specific growth factors and cell adhesion molecules. Interleukin 7 (IL-7), secreted by stromal cells, is an essential growth factor for early B cell development. IL-7 can stimulate pro and pre B cell proliferation [Duchosal, 1997]. Neutralizing anti-IL-7 antibody can inhibit IL-7 induced proliferation of pro- and pre-B cells [Duchosal, 1997]. IL-7 dependent pro-B cell proliferation is potentiated by insulin like growth factor-I and stem cell factor, two stromal growth factors [Duchosal, 1997]. Interferons (IFNs)-α/β, secreted by macrophages in bone marrow, can inhibit IL-7 induced B cell growth through apoptosis [Burrows, 1997]. The stromal cell-derived factor 1 or pre-B cell growth-stimulating factor (SDF-1/PBSF), produced constitutively by bone marrow stromal cells, stimulates proliferation of pro and pre-B cells [Nagasawa, 1996]. In vivo experiments show that mice lacking PBSF/SDF-1 died perinatally [Nagasawa, 1996]. IL-3 stimulates pre-B cell proliferation through the interaction with IL-3 receptor on B cells. [Duchosal, 1997]. IL-3 is a T cell derived cytokine and together with IL-6, can stimulate multipotential stem cells and B cell progenitor [Kincade, 1989]. Neuroleukin, a glucose-6-phosphate isomer homolog, has the ability to stimulate B cell development [Kincade, 1989].
There are several growth factors that negatively regulate B cell development. IL-1 inhibits generation of pre-B cells from earlier pro-B cells [Ryan, 1994]. However, IL-1 increases the generation of Ig secreting B cells from human bone marrow culture [Ryan, 1994]. TNF-α and IL-4 inhibit human lymphoid progenitor colonies [Ryan, 1994]. Cell adhesion molecules are also important for early B cell development. Stem cell factor (SCF), present on the cell surface of stromal cells, interacts with the cell-surface receptor tyrosine kinase, kit, on B cell precursor and stimulates early B cell development [Ashman, 1999]. SCF exists in both soluble and membrane bound form as a result of differential splicing and proteolytic cleavage [Ashman, 1999]. The membrane bound form of SCF contributes to its regulation of early B cell development [Ashman, 1999]. Flk2/flt3 is a receptor tyrosine kinase in the same family as the stem cell factor receptor c-kit. The flk2/flt3 ligand, which has homology to CSF-1, is a potent costimulator of early pro-B cells, in addition to IL-7 and SCF [Burrows, 1997]. Disruption of the flk2/flt3 gene leads to a selective deficiency of primitive B cell progenitors [Burrows, 1997].
VLA-4 is a cell surface molecule of B-cell precursors that interacts with the extracellular matrix ligand fibronectin on stromal cells and macrophages, and VCAM-1 on endothelial cells and macrophages [Duchosal, 1997]. VLA-4 expresses more on pro-B cells than pre B cells. Therefore, VLA-4 modulates pro-B cell proliferation more effectively than pre-B cells [Duchosal, 1997].
The interaction between CD44 on B-cell precursors and hyaluronate on stromal cells also plays an important role in B cell development. Antibodies to CD44 inhibit mouse B cell development in vivo [Duchosal, 1997]. Hormones may also regulate B lymphopoiesis. Estrogen regulates B cell generation via an effect on stromal cells in the lymphopoietic microenvironment [Burrows, 1997]. Dwarf mice deficient in the expression of prolactin and thyroid-stimulating hormone are immunodeficient, due to a T cell deficiency and a defect in B cell development, which is correctable by the lack of thyroid hormone thyroxine [Burrows, 1997].
T helper cells transmit signals to B cells through a direct contact of the B cell and the helper T cell. This direct contact is accomplished by antigen independent interaction of accessory molecule, CD40 ligands on the T helper cell and CD40s on the B cell help T cells [Parker, 1993], and by antigen-specific interaction of the peptide:MHC class II complex on the B cell surface with antigen-specific T cell receptor on helper T cells. Antigen-mediated B cell activation occurs in a T cell-independent mode or a T cell-dependent mode. T cell-independent activation of B cells can occur in response to non-protein antigen, such as a polysaccharide. The ability of B cells to respond directly to polysaccharides provides a rapid response to many important bacterial pathogens [Vos, 2000]. T cell-dependent activation of B cells takes place in response to protein-antigens or to non-protein antigens conjugated to protein carrier molecules.
T-cell dependent activation of B cells is the core of humoral immunity. Activated helper T cells produce soluble cytokines that can stimulate B cell proliferation and differentiation [Parker, 1993]. The first identified soluble cytokine was IL-4, also known as B-cell stimulatory factor 1 (BSF-1) or B cell growth factor (BCGF). IL-4 was originally identified as a molecule able to stimulate DNA synthesis of anti-IgM-stimulated murine B lymphocytes [Howard, 1982]. Human IL-4 is a 153 amino acid glycoprotein having a protein core with a molecular mass of 15 KD. Glycosylated human IL-4 can have a molecular mass of 20 KD [Yokota, 1986; Ohara, 1987]. IL-4 has multiple effects on B cells. For example, IL-4 can enhance the proliferation of B cells stimulated with anti-IgM antibody [Howard, 1982], induce the expression of class II MHC expression and CD23 [Conrad, 1987; Jansen, 1990], and regulate immunoglobulin isotype expression. For example, IL-4 is able to induce B cells to produce IgE [Pene, 1988] and induce the switching of expression of cells from producing IgM to IgG1 and/or IgE [Callard, 1991]. IL-4 also plays a role in the regulation of T cells, mast cells, monocytes, hematopoiesis, fibroblasts, and NK cells [Jansen, 1990].
Interleukin-2 (IL-2) is a 133 amino acid glycoprotein with a molecule weight of 13 to 17.5 KD according to viable glycosylation [Robb, 1981]. IL-2, produced by T cells, stimulates the proliferation of activated B cells [Gearing, 1985], promotes the induction of immunoglobulin secretion and J chain synthesis by B cells [Gearing, 1985; Blackman, 1986], and acts to enhance immune effects mediated by activated B cells [Mingari, 1984].
Interleukin 6 (IL-6) is a 186 amino acid glycoprotein with a molecular weight of 19 to 30 KD [May, 1989] that is produced from many cell types including monocytes, macrophages, stromal cells, and plasma cells [May, 1988; Frassanito, 2001]. IL-6 is well established as a late-stage differentiation factor for B cell to plasma cell transition [Muraguchi, 1988]. IL-6 stimulates activated B cells to produce IgM, IgG, and IgA [Muraguchi, 1988]. IL-6 also augments antigen-specific antibody response to antigen in vitro and in vivo [Takatsuki, 1988]. While the antigen-dependent phase of T cell development depends on the production of an autocrine factor, IL-2, a corresponding autocrine regulatory factor for B cells has not yet been identified.
PCDGF (PC-cell derived growth factor) is a highly tumorigenic autocrine growth factor and causative agent for a wide variety of tumors. For example, PCDGF levels are elevated in tumorigenic hematopoetic cells such as B cell leukemias, but cannot be detected in normal B cells. As described in U.S. Pat. No. 6,309,826, incorporated by reference herein in its entirety, overexpression of PCDGF leads to uncontrolled tumor cell growth and increased tumorigenesis. The degree of PCDGF overexpression directly correlates with the degree of cellular tumorigenicity. Cells overexpressing PCDGF do not require external signals to maintain uncontrolled cell growth. Loss of regulated cell growth, such as a loss in responsiveness to insulin and/or estrogen, leads to increased malignancy and excessive unregulated cell growth. However, PCDGF has not previously been shown to be associated with stimulating the growth of non-tumorigenic (i.e., normal) B cells.
While PCDGF moderately increases the growth of 3T3 cells, PCDGF inhibits the growth of several other cell, lines. For example, PCDGF inhibits the growth of normal mink lung epithelial cells (CCL 64) cells. Xia, X and Serrero, G, Identification of cell surface binding sites for PC cell derived growth factor, (epithelin/granulin precursor) on epithelial cells and fibroblasts. Biochem. Biophys. Res. Commun. 245, 539-543, 1998. PCDGF also inhibits the growth of normal mouse and rat thymic epithelial cells (BT1B and TEA3A1 cells) (Serrero, unpublished results).
PCDGF has no effect on the proliferation of several normal human cell lines including Hela and CHO cells (Serrero, unpublished data), and Cos-7 cells (Plowman, et al, 1993; Serrero unpublished results; Plowman, G. D., Green, J. M., Neubauer, M. G., Buckley, S. D., McDonald, V. L., Todaro, G. J., and Shoyab, M. (1992). The epithelin precursor encodes two proteins with opposing activities).